Making a Mightier Knockout Mouse

A regular C57BL/6 mouse with black fur (top) sits next to a C57BL/6 knockout mouse (bottom) easily identified by its reddish-brown fur

Somewhere in the world, there's someone working hard to build a better mouse trap.

In the meantime, a team supported with partial funding from the National Institutes of Health (NIH), has figured out how to build a better "knockout" mouse, a key research tool for exploring the genetic factors involved in health and disease.

Researchers from the Wellcome Trust Sanger Institute in Hinxton, England; the University of California, Davis; and Harvard University in Boston, have developed a new method for creating black 6 ES cells that produce black 6 knockout mice in a majority of cases, a strain of mice preferred by researchers because they have a similar genetic background to humans and can often mimic human health and disease. The team describes its pioneering achievement in the June 14 advanced online edition of Nature Methods.

In addition to NIH, the Wellcome Trust Sanger Institute and the Sixth Framework Programme of the European Union funded the research. Researchers carried out the work as part of the international Knockout Mouse Project, which is partly supported by NIH. Launched in 2006, the five-year project plans to build a comprehensive and publicly available resource of knockout mutations for each of the approximately 21,000 genes in the mouse genome.

First Knockout Mice

Researchers created the first knockout mice in 1989, using genetic engineering techniques to completely disrupt, or "knock out", one or more genes in the laboratory animals. Scientists use these mice to learn what the knocked out gene normally does, as well as what happens when its function is disrupted. Knockout mice have proven valuable for studying and modeling a wide range of human conditions, including obesity, heart disease, diabetes, arthritis, substance abuse, anxiety and aging.

But, despite those advances, researchers faced many limitations in what they could learn from knockout mice because the strain that was easiest to genetically engineer, called the 129 strain, is not the best mouse model for studying human biology. Most researchers prefer working with a strain of mouse called C57BL/6, commonly referred to as black 6. Used widely in scientific work for decades, black 6 mice are similar genetically to humans and often mimic human health and disease. It also was the same strain used to sequence the mouse genome. However, until now, researchers' have had little success with efficiently creating black 6 knockout mice.

Producing Knockout Mice

Researchers begin the process of making knockout mice by obtaining embryonic stem (ES) cells from early-stage mouse embryos. They use ES cells because these cells can differentiate into any type of adult cell. That means if a gene is knocked out in an ES cell, the effects can be observed in any tissue in an adult mouse.

To produce knockout mice, researchers then introduce an artificial piece of DNA into the nucleus of an ES cell grown in laboratory conditions. At each end of this DNA piece are base pairs that are identical in sequence, or homologous, to the base pairs that flank the mouse gene targeted for knock out. The cell's machinery automatically recognizes the identical stretches of DNA sequence and swaps out the existing mouse gene with the artificial piece of DNA. Because the artificial DNA is inactive, the swap knocks out the function of the mouse gene.

After a targeted gene is knocked out, researchers inject the altered ES cells into early-stage mouse embryos. The embryos are then implanted into the uterus of a female mouse and allowed to develop into mouse pups. Researchers screen the pups to find those with some tissues in which a gene is knocked out, along with some normal tissues from the embryos into which the altered ES cells were injected. Researchers then crossbreed the offspring of such mice to produce lines of mice in which both copies of the gene are knocked out in all tissues - a homozygous knockout.

Historically, scientists have created knockout mice with ES cells derived from the 129 strain of mice because these animals consistently transmitted knockout genes throughout the extensive breeding process needed to create a line of homozygous knockouts. In contrast, researchers found that ES cells from the black 6 strain of mice inexplicably quit working after a few generations.

A Better Knockout Mouse

Still, the quest to create black 6 knockout mice continued because the 129 mouse strain displays behavioral and physical defects that make them less than ideal for genetic studies involving neurobiology, immunology and physiology. In the past, researchers who wanted to produce a black 6 knockout mouse had to inject ES cells from 129 mice into early-stage mouse embryos and implant the embryos into a black 6 female mouse. Because the procedure involved two strains of mice, researchers had to breed 10 generations of mice to create a line of knockout mice with a pure black 6 genetic background. The entire process could take five years and cost a great deal of money.

To find which black 6 ES cells might work best for creating knockout mice, the scientists from the Knock Out Mouse Project experimented with ES cells from two substrains of black 6 called C57BL/6N and C57Bl/6J. First, they found that the black 6N ES cells, called JM8, could be grown more easily in the lab than the black 6J ES cells. Next, they tested the resulting black 6N ES cells by injecting them into 320 early-stage mouse embryos, and then implanting the embryos into female black 6 mice.

It worked. The procedure transferred the desired genetic material to the mouse pups at least 65 percent of the time.

"It's still a bit of a mystery why ES cells from black 6N proved to work so well in creating these knockout mice, when so many others had run into so much difficulty in the past," said Colin Fletcher, Ph.D., program director of the Knockout Mouse Project at the National Human Genome Research Institute, part of NIH. "Regardless, this line of cells appears to be up to the task of creating black 6 knockout mice at the scale needed to meet the goals of the Knockout Mouse Project."

The researchers also developed a simple and innovative way to sort the mouse pups produced by their procedure. Black 6 mice carry a genetic mutation that makes their coats black. The normal version of this gene produces a reddish-brown coat, called "agouti." Researchers restored the agouti gene in the black 6N ES cells and transplanted these ES cells into early-stage embryos from mice with a black coat color. The offspring of these mice have either black or agouti fur, allowing researchers to easily distinguish the pups with knocked out genetic material (agouti) from regular pups (black).

"This technique saves a breeding step and eliminates the need to do the extensive genetic testing on the tissues of mouse pups to determine whether they had inherited the knocked out genetic material," said Dr. Fletcher. "Thanks to this achievement, we can accelerate our efforts to use knockout mice to better understand human biology and improve human health."